Aspergillus fumigatus is the most common opportunistic hyphomycete, and invasive pulmonary aspergillosis caused by A. fumigatus continues to have high morbidity and mortality in neutropenic patients. The organism is a ubiquitous fungus that thrives in compost heaps where it is a major contributor to carbon and nitrogen recycling. In order to initiate infection, airborne conidia must survive a stressful transition from the compost heap to the mammalian lung. Temperature shift, change in nutritional availability, and attack by inimical host defenses are potent stressors to which the fungus must respond through environmental sensing and genetic and physiologic reprogramming. The protein kinase A (PKA) pathway has been identified in Saccharomyces as one of the primary signaling pathways involved in reprogramming in response to stress, however, little is known about the importance of this pathway in the growth and virulence of filamentous fungi. This proposal will test the hypothesis that intact signaling through PKA is required for induction of protective stress responses by A. fumigatus. This hypothesis will be tested through a series of four aims. In aim 1, deletion mutants of the regulatory and the catalytic subunit, and their respective complemented strains, will be tested in a murine model for virulence. The second aim will determine the extent to which mutants with unregulated PKA are susceptible to stressors such as oxidative damage, DMA damage, and extremes of pH. Aims 3 and 4 will address post-translational regulation of the PKA pathway in A. fumigatus. In aim 3, changes in phosphorylation patterns in the mutants will be used to define the PKA phosphoproteome. Phosphorylation state of the wild type following stress will aid in identifying stress-related targets of PKA phosphorylation. Compartmentalization plays a key role in eukaryotes in controlling the activity of PKA and some of its primary targets. Therefore, in aim 4, regulation of PKA activity will be examined by determining the subcellular localization of the PKA subunits of A. fumigatus under various nutritional and stress-induced states. The results of these studies should elucidate the role of PKA in the stress response of A. fumigatus and its influence on virulence.